The present invention is in the field of kinase proteins that are related to the calcium/calmodulin-dependent protein kinase subfamily, recombinant DNA molecules, and protein production. The present invention specifically provides novel peptides and proteins that effect protein phosphorylation and nucleic acid molecules encoding such peptide and protein molecules, all of which are useful in the development of human therapeutics and diagnostic compositions and methods.
Protein Kinases
Kinases regulate many different cell proliferation, differentiation, and signaling processes by adding phosphate groups to proteins. Uncontrolled signaling has been implicated in a variety of disease conditions including inflammation, cancer, arteriosclerosis, and psoriasis. Reversible protein phosphorylation is the main strategy for controlling activities of eukaryotic cells. It is estimated that more than 1000 of the 10,000 proteins active in a typical mammalian cell are phosphorylated. The high energy phosphate, which drives activation, is generally transferred from adenosine triphosphate molecules (ATP) to a particular protein by protein kinases and removed from that protein by protein phosphatases. Phosphorylation occurs in response to extracellular signals (hormones, neurotransmitters, growth and differentiation factors, etc), cell cycle checkpoints, and environmental or nutritional stresses and is roughly analogous to turning on a molecular switch. When the switch goes on, the appropriate protein kinase activates a metabolic enzyme, regulatory protein, receptor, cytoskeletal protein, ion channel or pump, or transcription factor.
The kinases comprise the largest known protein group, a superfamily of enzymes with widely varied functions and specificities. They are usually named after their substrate, their regulatory molecules, or some aspect of a mutant phenotype. With regard to substrates, the protein kinases may be roughly divided into two groups; those that phosphorylate tyrosine residues (protein tyrosine kinases, PTK) and those that phosphorylate serine or threonine residues (serine/threonine kinases, STK). A few protein kinases have dual specificity and phosphorylate threonine and tyrosine residues. Almost all kinases contain a similar 250-300 amino acid catalytic domain. The N-terminal domain, which contains subdomains I-IV, generally folds into a two-lobed structure, which binds and orients the ATP (or GTP) donor molecule. The larger C terminal lobe, which contains subdomains VI A-XI, binds the protein substrate and carries out the transfer of the gamma phosphate from ATP to the hydroxyl group of a serine, threonine, or tyrosine residue. Subdomain V spans the two lobes.
The kinases may be categorized into families by the different amino acid sequences (generally between 5 and 100 residues) located on either side of, or inserted into loops of, the kinase domain. These added amino acid sequences allow the regulation of each kinase as it recognizes and interacts with its target protein. The primary structure of the kinase domains is conserved and can be further subdivided into 11 subdomains. Each of the 11 subdomains contains specific residues and motifs or patterns of amino acids that are characteristic of that subdomain and are highly conserved (Hardie, G. and Hanks, S. (1995) The Protein Kinase Facts Books, Vol I:7-20 Academic Press, San Diego, Calif.).
The second messenger dependent protein kinases primarily mediate the effects of second messengers such as cyclic AMP (cAMP), cyclic GMP, inositol triphosphate, phosphatidylinositol, 3,4,5-triphosphate, cyclic-ADPribose, arachidonic acid, diacylglycerol and calcium-calmodulin. The cyclic-AMP dependent protein kinases (PKA) are important members of the STK family. Cyclic-AMP is an intracellular mediator of hormone action in all prokaryotic and animal cells that have been studied. Such hormone-induced cellular responses include thyroid hormone secretion, cortisol secretion, progesterone secretion, glycogen breakdown, bone resorption, and regulation of heart rate and force of heart muscle contraction. PKA is found in all animal cells and is thought to account for the effects of cyclic-AMP in most of these cells. Altered PKA expression is implicated in a variety of disorders and diseases including cancer, thyroid disorders, diabetes, atherosclerosis, and cardiovascular disease (Isselbacher, K. J. et al. (1994) Harrison""s Principles of Internal Medicine, McGraw-Hill, New York, N.Y., pp. 416-431, 1887).
Calcium-calmodulin (CaM) dependent protein kinases are also members of STK family. Calmodulin is a calcium receptor that mediates many calcium regulated processes by binding to target proteins in response to the binding of calcium. The principle target protein in these processes is CaM dependent protein kinases. CaM-kinases are involved in regulation of smooth muscle contraction (MLC kinase), glycogen breakdown (phosphorylase kinase), and neurotransmission (CaM kinase I and CaM kinase II). CaM kinase I phosphorylates a variety of substrates including the neurotransmitter related proteins synapsin I and II, the gene transcription regulator, CREB, and the cystic fibrosis conductance regulator protein, CFTR (Haribabu, B. et al. (1995) EMBO Journal 14:3679-86). CaM II kinase also phosphorylates synapsin at different sites, and controls the synthesis of catecholamines in the brain through phosphorylation and activation of tyrosine hydroxylase. Many of the CaM kinases are activated by phosphorylation in addition to binding to CaM. The kinase may autophosphorylate itself, or be phosphorylated by another kinase as part of a xe2x80x9ckinase cascadexe2x80x9d.
Another ligand-activated protein kinase is 5xe2x80x2-AMP-activated protein kinase (AMPK) (Gao, G. et al. (1996) J. Biol Chem. 15:8675-81). Mammalian AMPK is a regulator of fatty acid and sterol synthesis through phosphorylation of the enzymes acetyl-CoA carboxylase and hydroxymethylglutaryl-CoA reductase and mediates responses of these pathways to cellular stresses such as heat shock and depletion of glucose and ATP. AMPK is a heterotrimeric complex comprised of a catalytic alpha subunit and two non-catalytic beta and gamma subunits that are believed to regulate the activity of the alpha subunit. Subunits of AMPK have a much wider distribution in non-lipogenic tissues such as brain, heart, spleen, and lung than expected. This distribution suggests that its role may extend beyond regulation of lipid metabolism alone.
The mitogen-activated protein kinases (MAP) are also members of the STK family. MAP kinases also regulate intracellular signaling pathways. They mediate signal transduction from the cell surface to the nucleus via phosphorylation cascades. Several subgroups have been identified, and each manifests different substrate specificities and responds to distinct extracellular stimuli (Egan, S. E. and Weinberg, R. A. (1993) Nature 365:781-783). MAP kinase signaling pathways are present in mammalian cells as well as in yeast. The extracellular stimuli that activate mammalian pathways include epidermal growth factor (EGF), ultraviolet light, hyperosmolar medium, heat shock, endotoxic lipopolysaccharide (LPS), and pro-inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1).
PRK (proliferation-related kinase) is a serum/cytokine inducible STK that is involved in regulation of the cell cycle and cell proliferation in human megakaroytic cells (Li, B. et al. (1996) J. Biol. Chem. 271:19402-8). PRK is related to the polo (derived from humans polo gene) family of STKs implicated in cell division. PRK is downregulated in lung tumor tissue and may be a proto-oncogene whose deregulated expression in normal tissue leads to oncogenic transformation. Altered MAP kinase expression is implicated in a variety of disease conditions including cancer, inflammation, immune disorders, and disorders affecting growth and development.
The cyclin-dependent protein kinases (CDKs) are another group of STKs that control the progression of cells through the cell cycle. Cyclins are small regulatory proteins that act by binding to and activating CDKs that then trigger various phases of the cell cycle by phosphorylating and activating selected proteins involved in the mitotic process. CDKs are unique in that they require multiple inputs to become activated. In addition to the binding of cyclin, CDK activation requires the phosphorylation of a specific threonine residue and the dephosphorylation of a specific tyrosine residue.
Protein tyrosine kinases, PTKs, specifically phosphorylate tyrosine residues on their target proteins and may be divided into transmembrane, receptor PTKs and nontransmembrane, non-receptor PTKs. Transmembrane protein-tyrosine kinases are receptors for most growth factors. Binding of growth factor to the receptor activates the transfer of a phosphate group from ATP to selected tyrosine side chains of the receptor and other specific proteins. Growth factors (GF) associated with receptor PTKs include; epidermal GF, platelet-derived GF, fibroblast GF, hepatocyte GF, insulin and insulin-like GFs, nerve GF, vascular endothelial GF, and macrophage colony stimulating factor.
Non-receptor PTKs lack transmembrane regions and, instead, form complexes with the intracellular regions of cell surface receptors. Such receptors that function through non-receptor PTKs include those for cytokines, hormones (growth hormone and prolactin) and antigen-specific receptors on T and B lymphocytes.
Many of these PTKs were first identified as the products of mutant oncogenes in cancer cells where their activation was no longer subject to normal cellular controls. In fact, about one third of the known oncogenes encode PTKs, and it is well known that cellular transformation (oncogenesis) is often accompanied by increased tyrosine phosphorylation activity (Carbonneau H and Tonks N K (1992) Annu. Rev. Cell. Biol. 8:463-93). Regulation of PTK activity may therefore be an important strategy in controlling some types of cancer.
Calcium/Calmodulin-Dependent Protein Kinases
The novel human protein, and encoding gene, provided by the present invention is related to the family of calcium/calmodulin-dependent protein kinases, which are serine/threonine kinases. The protein of the present invention shows a high degree of similarity to calcium/calmodulin-dependent protein kinase II (CaM II), and the CaM II beta subunit in particular. Furthermore, the protein/cDNA of the present invention may be an alternative splice form of a protein provided in Genbank gi5326757 (see the amino acid sequence alignment in FIG. 2).
CaM II is comprised of alpha, beta, gamma, and delta subunits. Each subunit is encoded by a separate gene and alternatively splice forms of each subunit have been found (Breen et al., Biochem. Biophys. Res. Commun. 236 (2), 473-478 (1997)). CaM II exerts important effects on hormones and neurotransmitters that utilize calcium as a second messenger and has been implicated in a wide variety of neuronal and non-neuronal functions, including cell growth control (Tombes et al., Biochim Biophys Acta Mar. 1, 1997;1355(3):281-92). It has been found that certain CaM II isozymes are preferentially expressed in tumor cells and thus certain tumor cells express a completely different spectrum of CaM II isozymes compared with normal cells/tissues (Tombes et al., Biochim Biophys Acta Mar. 1, 1997;1355(3):281-92). Therefore, CaM II plays a key role in cell growth control and tumor proliferation and, importantly, novel human CaM II variants are valuable as potential diagnostic markers and therapeutic targets for cancer.
Expression of CaM II beta mRNA is elevated in the frontal cortex in schizophrenia and CaM II is known to play a key role in the amplified action of amphetamine induced-dopamine release, which is observed in schizophrenics (Novak et al., Brain Res. Mol. Brain Res. 82 (1-2), 95-100 (2000)). Thus, CaM II, and CaM II beta in particular, may play important roles in schizophrenia.
Beta-cell CaM II activity is associated with insulin secretion, and multiple isoforms of CaM II are expressed in human islets of Langerhans (Breen et al., Biochem. Biophys. Res. Commun. 236 (2), 473-478 (1997)). It has been suggested that CaM II controls activation-induced cellular differentiation, and is important for imparting antigen-dependent memory to T cells (Bui et al., Cell 100: 457-467, 2000). For a further review of CaM II and CaM II beta, see Wang et al., FEBS Lett. 475 (2), 107-110 (2000) and Li et al., Cytogenet. Cell Genet. 66: 113-116, 1994.
Calmodulin is a major Ca(2+)-binding protein in the brain, where it modulates numerous Ca(2+)-dependent enzymes and cellular functions. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is particularly important in the brain and is involved in a variety of neuronal functions (Sola et al., Prog Neurobiol 1999 Jun;58(3):207-32), such as postsynaptic responses (such as long-term potentiation), neurotransmitter synthesis and exocytosis, cytoskeletal interactions and gene transcription (Colbran, Neurochem Int December 1992;21(4):469-97). Ca2+ and calmodulin antagonists inhibit seizures induced by convulsant agents, indicating that the Ca2+/calmodulin signaling system plays an important role in the onset of seizures. Changes in CaMKII expression has been observed following seizures and, furthermore, expression of calmodulin and CaMKII in microglial cells in the brain increases following seizures (Sola et al., Prog Neurobiol June 1999;58(3):207-32). CaMKII levels are also altered in pathological states such as Alzheimer""s disease and ischemia (Colbran, Neurochem Int December 1992;21(4):469-97), suggesting a role of CaMKII in these disorders.
Calmodulin is also important for regulating the plasma membrane calcium pump, which transports Ca2+ out of cells. The pump is inactive in the absence of calmodulin, but is activated by calmodulin binding (Penniston et al., J Membr Biol Sep. 15, 1998;165(2):101-9).
Due to their importance in cell growth control, novel human CaM II proteins/genes, such as provided by the present invention, are valuable as potential targets for the development of therapeutics to treat cancer and other disorders. Furthermore, SNPs in CaM II genes, such as provided by the present invention, may serve as valuable markers for the diagnosis, prognosis, prevention, and/or treatment of cancer and other disorders.
Using the information provided by the present invention, reagents such as probes/primers for detecting the SNPs or the expression of the protein/gene provided herein may be readily developed and, if desired, incorporated into kit formats such as nucleic acid arrays, primer extension reactions coupled with mass spec detection (for SNP detection), or TaqMan PCR assays (Applied Biosystems, Foster City, Calif.).
Kinase proteins, particularly members of the calcium/calmodulin-dependent protein kinase subfamily, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown members of this subfamily of kinase proteins. The present invention advances the state of the art by providing previously unidentified human kinase proteins that have homology to members of the calcium/calmodulin-dependent protein kinase subfamily.
The present invention is based in part on the identification of amino acid sequences of human kinase peptides and proteins that are related to the calcium/calmodulin-dependent protein kinase subfamily, as well as allelic variants and other mammalian orthologs thereof. These unique peptide sequences, and nucleic acid sequences that encode these peptides, can be used as models for the development of human therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of human therapeutic agents that modulate kinase activity in cells and tissues that express the kinase. Experimental data as provided in FIG. 1 indicates expression in fetal brain, testis, lung small cell carcinoma, and uterus endometrium adenocarcinoma.